The long term objectives of the applicant's program are the elucidation of the chemical mechanism of action of thiamin diphosphate dependent enzymes, specifically two that decarboxylate pyruvic acid: pyruvate decarboxylase (EC 4.1.1.1) and the pyruvate dehydrogenase multienzyme complex. The latter enzyme is a key junction point in metabolism and its thorough understanding is of fundamental biomedical importance. 1. Potential mechanism-based irreversible inhibitors are being synthesized to covalently label the catalytic centers to enable sequencing of these centers. The primary sequences of the cofactor and substrate binding sites will then be compared in search for homologies in structures whose function is parallel. The primary sequences of these enzymes have been determined from their gene structures. 2. The structures of key enzyme-bound intermediates are mimicked synthetically to define their UV-VIS spectral characteristics. With the spectral information from the model compounds, the enzyme-bound intermediates will be identified by pre-steady state repaid kinetic means. The rates of: key elementary steps will be directly determined, including that of: the reductive acetyl transfer in pyruvate dehydrogenase. 3. Multinuclear Magnetic Resonance studies will be performed to study the environment, states of ionization, tautomerization and the conformation of the enzyme-bound thiamin diphosphate by employing specifically labelled (13C and 15N) coenzymes. The large quantities of enzymes needed will be produced by immuno affinity chromatographic means especially developed for this purpose.